Download The role and source of 5P-deoxyadenosyl radical in a carbon

FEBS 21016
FEBS Letters 437 (1998) 309^312
The role and source of 5P-deoxyadenosyl radical in a carbon skeleton
rearrangement catalyzed by a plant enzyme
Sandrine Ollagnier, Eric Kervio, Jaènos Reètey*
Lehrstuhl fuër Biochemie, Institut fuër Organische Chemie, Universitaët Karlsruhe, Kaiserstr. 12, D-76128 Karlsruhe, Germany
Received 15 August 1998; received in revised form 24 September 1998
Abstract The last step in the biosynthesis of tropane alkaloids
is the carbon skeleton rearrangement of littorine to hyoscyamine.
The reaction is catalyzed by a cell-free extract prepared from
cultured hairy roots of Datura stramonium. Adenosylmethionine
stimulated the rearrangement 10^20-fold and showed saturation
kinetics with an apparent Km of 25 WM. It is proposed that
S-adenosylmethionine is the source of a 5P-deoxyadenosyl radical
which initiates the rearrangement in a similar manner as it does
in analogous rearrangements catalyzed by coenzyme B12 dependent enzymes. Possible roles of S-adenosylmethionine as
a radical source in higher plants are discussed.
z 1998 Federation of European Biochemical Societies.
Key words: Tropane alkaloid; Carbon skeleton
rearrangement; S-Adenosylmethionine as 5P-deoxyadenosyl
radical donor
1. Introduction
The last and most intriguing step in the biosynthesis of the
tropane alkaloids hyoscyamine and scopolamine is the carbon
skeleton rearrangement depicted in Scheme 1 (for a review see
[1]). Due to its similarity to the rearrangement of methylmalonyl CoA to succinyl-CoA it was originally speculated that
coenzyme B12 is involved in the reaction [2^4]. Since, in spite
of some claims [5^7], no trace of vitamin B12 has ever been
found in plants this idea was abandoned [8,9]. More recently
it has been surmised that cytochrome P450 is the agent abstracting the benzylic hydrogen atom and redonating a hydroxyl group after the rearrangement [10,11]. Accordingly,
clotrimazole, a known P-450 inhibitor, has been said to inhibit
the formation of hyoscyamine [1], and the 3-HRe atom abstracted from the benzylic position was lost during the biosynthetic experiment [12]. When the OH group of littorine was
labelled with the isotope 18 O, 25^29% of the label was lost in
the product [11]. These results were interpreted as the involvement of two enzymes, a mutase and a dehydrogenase, in the
conversion of littorine into hyoscyamine, the geminal diol
being an intermediate in the reaction. The dehydratation
thereof to the aldehyde could be partially stereospeci¢c explaining the less than 50% loss of 18 O in the overall reaction.
Only a few agents are known in biochemistry that are able to
abstract hydrogen atoms from non-activated positions. Beside
cytochrome P450, nature uses the 5P-deoxyadenosyl radical
for this purpose. In animals and bacteria coenzyme B12 is
*Corresponding author. Fax: (49) (721) 608-4823.
E-mail: [email protected]
Abbreviations: SAM, S-adenosylmethionine
the major source of 5P-deoxyadenosyl radical [13]. More recently, S-adenosylmethionine (SAM) was also identi¢ed to
play a similar role [14]. Therefore, SAM has been called the
poor man's B12 [15].
Here we report on the involvement of SAM in the rearrangement of littorine to hyoscyamine.
2. Materials and methods
2.1. Chemicals
They were obtained from commercial sources and were the highest
purity normally available. SAM and [2,8,5P-3 H]ATP (65% 3 H in position 5P) were obtained from Amersham, Aylesbury, UK, dithiothreitol
(DTT) from Promega, ethylene diamine tetraacetic acid (EDTA) from
Serva, B5 medium from Bioproduct (Boehringer Ingelheim).
2.2. Preparation of the phenyl-lactoyl tropine (littorine)
According to a modi¢ed procedure [16] L-(3)-phenyllactic acid
(Fluka) (116 mg, 0.7 mmol) dried over phosphorus pentoxide was
added to tropine (Acros) (0.5 mmol) previously dried over sodium
hydroxide pellets. The mixture was heated to 145³C on an oil bath
and dry hydrogen chloride was bubbled through the solution for 3.5 h.
(Note that esteri¢cation attempts with para-toluene sulfonic acid or
under montmorrillonite KSF as alternative procedures gave no satisfactory results.) The cooled mixture was dissolved in a 10 ml H2 SO4
solution (50 mM), ¢ltered and made alkaline with a 10% aqueous
ammonium hydroxide solution, then extracted into chloroform
(6U10 ml). The solvent was removed under reduced pressure and
the residue puri¢ed by partition chromatography on Celite (Merck)
containing 0.5 M phosphate bu¡er (pH 6.8) eluted with chloroform.
GC-MS analysis of the combined fractions (97 mg, yield: 48%) indicated almost pure (98%) littorine.
Retention time (1,4-dioxane) on a Optima 1-02 capillary column
(25 mU0.2 mm), ramp 100^300³C at 20³C/min): 12.2 min. MS m/z
(rel. int.): 361(4), 193(3), 140(4), 125(12), 124(100), 123(1), 96(9),
95(5), 94(12), 91(8), 83(15), 73(19) [17].
13
C NMR and 1 H NMR spectra were recorded on a Bruker DRX
500 spectrometer. Deuteriochloroform was used as the solvent with
TMS as an internal reference for 1 H. The abbreviations used are as
follows: N, shift; s, singlet; d, doublet; t, triplet; m, multiplet; b,
broad.
1
H NMR (CDCl3 ) N: 1.65 (1H, H6 or H7 , d, J3 HH = 15.8 Hz); 1.85
(1H, H7 or H6 , d, J3 HH = 15.8 Hz); 1.94^2.20 (2H, H6 or H7 , m); 2.00
(N-CH3 , s); 2.54^2.72 (4H, H4 and H2 , m); 2.78 (1H, H5 or H1 , t,
J3 HH = 15.8 Hz); 2.80 (1H, H1 or H5 , t, J3 HH = 15.8 Hz); 2.98 (2H,
H30 , d, J3 HH = 6.5 Hz); 3.57 (b, OH); 4.37 (1H, H20 , t, J3 HH = 6.5 Hz);
4.99 (1H, H3 , t, J3 HH = 4.5 Hz); 7.11^7.22 (5Harom , m) [18]. 13 C NMR
(CDCl3 ) N: 25.1, 25.3 (C7 and C6 ); 35.1, 35.2 (C4 and C2 ); 37.3 (C30 );
40.7 (CH3 -N); 60.0 (C1 and C5 ); 71.6 (C3 ); 74.3 (C20 ); 126.9 (C70 );
128.5, 129.3 (C50 ;60 ;80 ;90 ); 136.4 (C40 ); 173.5 (C10 ).
2.3. Preparation of the SAM synthetase
SAM synthetase was puri¢ed by the procedure of Markham et al.
[19], with some minor modi¢cations: growth in the presence of oxytetracycline (30 Wg/ml) and disruption of the cells by sonication (Bandelin Nonopuls HD 2200). The speci¢c activity of the enzyme was
150 000 pmol/min/mg.
2.4. Preparation of the S-[2,8,5P-3 H]adenosylmethionine
S-[2,8,5P-3 H]Adenosylmethionine was enzymatically synthesized
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S. Ollagnier et al./FEBS Letters 437 (1998) 309^312
from [2,8,5P-3 H]ATP (speci¢c activity: 41 mCi/mmol) and unlabelled
methionine, using a SAM synthetase preparation.
The reaction mixture contained 200 Wl of 0.1 M diluted solution of
[2,8,5P-3 H]ATP (speci¢c activity: 424 700 cpm/nmol) in 3 ml Tris-HCl
bu¡er (pH 7.4), adjusted to pH 7,6 by addition of a 2 M KOH
solution, 1.5 eq. MgCl2 , 5 eq. KCl, 1.2 eq. L-methionine added as
solids, 100 Wl reduced glutathione (25 mg/ml) and 11 mg of SAM
synthetase were incubated for 45 min at 35³C. The reaction was terminated by addition of 40 Wl of trichloroacetic acid (6 M). The resulting suspension was centrifuged at 4³C and the extracts pooled and
stored at 4³C. After lyophilization, the residue was dissolved in
doubly distilled water and applied to a carboxymethyl sepharose
high £ow column (0.8U20 cm FPLC column, Pharmacia Biotech,
Freiburg, Germany) previously equilibrated with water. The column
was washed with 2 volumes of water and then S-[2,8,5P3
H]adenosylmethionine was eluted with 1 M NaCl gradient (4 ml/
min). Fractions containing S-[2,8,5P-3 H]adenosylmethionine (speci¢c
activity: 123 000 cpm/nmol) were mixed, lyophilized and stored at
320³C until used.
2.5. Plant material
Roots from Datura stramonium D15/5 were a gift from Dr. N.J.
Walton (Institute of Food Research, Norwich, UK). Roots cultures
were initiated by wounding surface sterilized explants of leaves on the
midrib with an overnight suspension of Agrobacterium rhizogenes.
Tissue bearing emergent roots were excised and placed into 8 ml of
Gamborg's B5 medium supplemented by 500 Wg/ml ampicillin. Roots
for enzyme extraction and puri¢cation were grown at 25³C, under
stirring at 90 rpm. Rapidly growing roots were subcultured every
2 weeks into 50 ml of the same medium in a 250 ml Erlenmeyer £ask.
After eight subcultures it should be possible to omit ampicillin from
the medium. After this time, roots were harvested by ¢ltration in
vacuo, washed twice with water and frozen immediately at 370³C.
2.6. Protein extraction
All procedures were done at 4³C. Roots (6 g) were crushed in a
mortar with sea sand, mixed with 15 ml of extraction bu¡er (100 mM
K2 HPO4 pH 8, 3 mM DTT, 5 mM Na2 EDTA and 250 mM sucrose)
and then ground during 30 min until a homogeneous suspension was
obtained. The homogenate was clari¢ed by passage through Miracloth (Calbiochem, Novabiochem, Frankfurt a/M, Germany) and centrifugation (30 000Ug, 4³C, 30 min). Low molecular weight compounds were removed using a PD-10 desalting column (Pharmacia
Biotech AB, Uppsala, Sweden) equilibrated in a one tenth strength
extraction bu¡er. The proteins were eluted with 3.5 ml of the same
bu¡er. The yellow solution obtained was concentrated to 2^4 mg/ml
with centricon 5000 Da (Pharmacia Biotech AB, Uppsala, Sweden).
2.7. Enzyme assay
The activity of the enzyme was determined by measuring the conversion of littorine into hyoscyamine by the GC-MS method. The
standard reaction mixture, in a volume of 200 Wl, contained 200 WM
SAM (unlabelled or labelled as prepared above), 100 Wl of the protein
solution (200^400 Wg), 2 mM of littorine, adjusted to 200 Wl with the
extraction bu¡er. After 40 min incubation at 33³C, the reaction was
terminated by addition of 100 Wl 30% NH4 OH. Control experiments
lacking substrate were routinely included. Reaction mixtures were
loaded on an Extrelut-1 column (Merck Art 1.13076, Darmstadt,
Germany). After 15 min the alkaloids were eluted with 13 ml of
CHCl3 . The chloroform fractions were evaporated to dryness at
35³C, dissolved in 200 Wl of a fresh solution of 1,4 dioxane/N,O-bis(trimethylsilyl)-acetamide (4:1 v/v), analyzed by GC-MS (experiments
with unlabelled SAM) and HPLC.
2.8. Assay of protein
Protein was assayed by the method of Smith et al. [20] using com-
mercially prepared reagent (bicinchoninic acid) and bovine serum albumin as standard.
2.9. Extraction and analysis of alkaloids
Harvested roots were freeze-dried, then powdered and soaked overnight in an EtOH-28% NH4 OH (19:1) mixture. This macerated material was centrifuged for 5 min at 1300Ug. Extraction with the basic
alcohol was repeated twice and the combined alcohol fractions were
evaporated to dryness at 35³C. The dry residue was dissolved in 2 ml
of 0.1 N HCl and the acidic aqueous solution was ¢ltered through
¢lter No. 2. One milliliter of the ¢ltrate was made alkaline with 2 ml
of 1 M Na2 CO3 -NaHCO3 bu¡er (pH 10) and 1 ml of this alkaline
solution was loaded onto a Extrelut-1 column. After 10 min, 6 ml of
CHCl3 was passed through the column and the chloroform fractions
were evaporated to dryness at 35³C. The dry residue was dissolved in
a mixture of 1,4 dioxane/N,O-bis-(trimethylsilyl)-acetamide (v/v 4:1).
Alkaloids were measured with a gas chromatograph coupled with a
mass spectrometer model Hewlett Packard 5890 Serie II using a capillary column Optima 1-02 Wm (25 mU0.2 mm). The column temperature was 350³C, the carrier gas was H2 at a £ow rate of 20 ml/min.
The detector FID were used.
The two alkaloids identi¢ed for this study were littorine and hyoscyamine [17].
Littorine RT (retention time): 12.2 min. MS m/z (rel. int.): 361(4),
193(3), 140(4), 125(12), 124(100), 123(1), 96(9), 95(5), 94(12), 91(8),
83(15), 73(19).
Hyoscyamine RT: 12.1 min. MS m/z (rel. int.): 361(5), 193(0.5),
140(7), 125(10), 124(100), 123(6), 104(6), 96(10), 95(6), 94(13),
83(20), 82(18), 73(15).
2.10. Reverse phase HPLC chromatography
An Incapharm 100 RP-18 TS (250U4.6 mm) column was equilibrated at room temperature with 0.1% tri£uoroacetic acid in water
solution. 5 min after sample injection (20 Wl in same bu¡er), a gradient of 0^60% solvent B (0.1% tri£uoroacetic acid in acetonitrile) in
40 min was developed by a Hewlett-Packard 1050 T system at a £ow
rate of 1.2 ml/min. The eluted substances were monitored at 215 and
254 nm. Labelled products were analyzed by liquid scintillation counting (Tri-carb 2100TR, Packard, a Canberra company, Meriben, USA)
after adding 15 ml of scintillation liquid (Lumasafe plus^Lumac LSC)
to the peak fractions.
2.11. Determination of the isotope exchange
Tritium exchange with water was measured by liquid scintillation counting (Tri-carb 2100 TR) after loading 10 ml of scintillation
liquid to 10 Wl of water resulting from bulb-to-bulb distillations. The
di¡erence in emission was measured between S-[2,8,5P3
H]adenosylmethionine solution (speci¢c activity: 123 000 cpm/nmol)
and the resulting distilled water and between aqueous phase (0.1%
tri£uoroacetic acid in doubly distilled water) before HPLC analysis
and the resulting distilled water.
3. Results
3.1. Dependence of the rearrangement littorine/hyoscyamine on
SAM
The experiments were carried out as described in Section
2.7 and are listed in Table 1. The silylated products were
detected by GLC coupled with MS. Omission of SAM from
the assay mixture resulted in only 3.5% hyoscyamine while
96.5% remained littorine. Addition of SAM (200 WM) increased the conversion leading to 61% hyoscyamine. When
the hairy root extract was kept frozen at 320³C for a week
Table 1
Dependence of the rearrangement of tropane alkaloids on SAM
Hairy roots
SAM (WM)
% GC-MS hyoscyamine
% GC-MS littorine
Fresh extract
Fresh extract
One freeze-thawing
One freeze-thawing
0
200
0
200
3.5
61
2.3
28
96.5
39
97.7
72
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S. Ollagnier et al./FEBS Letters 437 (1998) 309^312
311
4. Discussion
Scheme 1. Carbon skeleton rearrangement of tropane alkaloids.
the conversion under the same conditions without and with
SAM decreased to 2.3 and 28% hyoscyamine, respectively.
After 4 weeks of freezing little if any mutase activity of the
preparation could be detected.
3.2. Determination of the Michaelis constant for SAM
As the enzyme source we used a hairy root extract, thawed
after 1 week in the deep-freeze. The kinetic experiments were
carried out under conditions of the enzyme assay (see Section
2.7), except that the concentration of SAM was varied between 50 and 500 WM. The Km and Vmax values were determined using Lineweaver-Burk plot (Fig. 1).
The following values were found: Km = 25 þ 5 WM, Vmax =
29 Wmol/40 min.
3.3. Conducting the rearrangement in the presence of
S-[2,8,5P-3 H]adenosylmethionine
When the enzymic rearrangement of littorine to hyoscyamine was carried out in the presence of [2,8,5P-3 H]SAM
(see Section 2.4 for preparation), no incorporation of tritium
into the alkaloids was observed. However, about 2% of the
tritium (calculated for the 5P position) was washed out into
the medium. Only a trace of tritium was found in the solvent
in a control experiment without hairy root extract. HPLC
showed no appreciable amounts of 5P-deoxyadenosine after
the reaction. If the latter was an intermediate, then only in
enzyme-bound catalytic amounts. If the putative 5P-deoxyadenosyl radical abstracted the benzylic pro-R H-atom, then this
process must have been irreversible. Possible reasons for
the loss of tritium from the 5P position of SAM are given
below.
The 10^20-fold enhancement of the carbon skeleton rearrangement, littorine to hyoscyamine (Scheme 1) by SAM in
cell-free extracts of a root culture of Datura stramonium suggests a new role for this cofactor in plant biochemistry. The
well-known function of SAM as methyl donor is operative in
both prokaryotic and eukaryotic organisms and requires a
heterolytic cleavage of a carbon sulfur bond. More recently,
Frey and coworkers discovered and documented [14,15] a case
in which SAM is the source of a 5P-deoxyadenosyl radical, the
homolytic cleavage of the carbon sulfur bond being promoted
reductively by an Fe4 S4 cluster-containing enzyme.
We surmise a similar role for SAM in the rearrangement of
littorine to hyoscyamine. Indeed, addition of SAM to the
assay mixture not only increased the conversion of littorine
to hyoscyamine, but showed saturation kinetics with a Km of
25 WM (Fig. 1).
The low activity of the hairy root extracts without addition
of SAM may be due to trace amounts of endogenous SAM
that remained in the extract after the PD-10 desalting column.
Indeed, in previous studies no SAM was added to the cell-free
system, but much more extract and longer incubation times
were used. Moreover, most of the incorporation experiments
were carried out with growing root cultures and the incubation times were 10^28 days. In these cases endogenous SAM
must have been provided by the in vivo system.
Preliminary experiments using [2,8,5P-3 H]SAM showed no
incorporation of tritium into littorine or hyoscyamine but
some loss of the radioactivity to water. This is in agreement
with the observed loss of the benzylic pro-R H-atom of littorine during the rearrangement [12]. Since all work has been
done with root cultures or crude cell-free extracts it is possible
that the migrated H-atom is washed out by redox enzymes.
This could also explain the 25^29% loss of 18 O of littorine
during the rearrangement [11] since the intermediate aldehyde
could be in equilibrium with its hydrate.
The steric course of the migration at the relevant centers is
also of importance. After contradictory results [2,3,21] it has
been established that the substitution occurs with inversion at
both migration centers [12]. The same steric course has been
determined for the lysine 2,3-aminomutase reaction [22]. In
the latter, however, the migrating hydrogen atom is not lost
Fig. 1. Lineweaver-Burk determination of Km and Vmax . X = % GC-MS hyoscyamine.
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to the medium. This discrepancy can only be clari¢ed when
pure littorine mutase is available. Another plant enzyme that
catalyzes the rearrangement of K-phenylalanine to L-phenylalanine is the phenylalanine aminomutase. This reaction is
part of taxol biosynthesis. Recently it has been established
that the substitution occurs with retention of con¢guration
at both migration centers [23]. This fact does not rule out
the possibility that the same cofactor, namely SAM, is operative also in the phenylalanine aminomutase reaction. Precedence for opposite steric courses is found in the coenzyme
B12 -dependent carbon skeleton rearrangements. Methylmalonyl-CoA mutase operates with retention [24,25], methylene
glutarate mutase [26] and glutamate mutases [27] with inversion of con¢guration. Even the same enzyme, the coenzyme
B12 -dependent ethanolamine ammonia-lyase, operates with
opposite steric courses, inversion and retention with (2R)and (2S)-propanolamines, respectively [28]. We thus propose
that SAM may be the cofactor in the B12 -like rearrangements
occurring in plants and the reaction is initiated by the 5Pdeoxyadenosyl radical derived from SAM.
That plants use SAM as the source of 5P-deoxyadenosyl
radical has been established for the last step of biotin biosynthesis in Arabidopsis thaliana [29]. A similar mechanism operates in Bacillus sphaericus and Escherichia coli [30,31]. We
thus believe that while bacteria are able to use both coenzyme
B12 and SAM as sources of 5P-deoxyadenosyl radical, animals
and humans inherited from them the former and higher plants
the latter.
Acknowledgements: The work was supported by the European Union
and the Fonds der Chemischen Industrie. We thank Dr. Nick Walton,
Institute of Food Research, Norwich, UK, and Professor Meinhart
Zenk, University of Munich, Germany, for help with establishing the
hairy root culture.
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